Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-03-03
2001-07-17
Jones, W. Gary (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S005000, C435S006120, C435S091100, C435S091200, C427S007000, C427S145000, C427S337000, C427S338000, C283S073000, C283S074000, C283S095000, C283S098000, C283S100000, C118S031500, C118S201000
Reexamination Certificate
active
06261809
ABSTRACT:
The invention relates to a process according to the precharacterizing clause of marking a solid, liquid, or gaseous substance. It furthermore relates to a kit for carrying out the process.
Such a process is disclosed in U.S. Pat. No. 5,643,728. In this process, the marking is contained in a specific sequence section of a nucleic acid sequence. For identification of the marking, the nucleic acid sequence is duplicated using the polymerase chain reaction (PCR). The marking containing [sic] in the duplicated product is then identified by means of sequencing.
A further process is disclosed in DE 44 39 896 A1. In this process, the DNA molecules added to the substance to be marked accommodate a linear polymorphism which carries the marking. For identification of the marking, the DNA molecules are duplicated by means of PCR and then subjected to gel electrophoresis. The band sequence which can be observed as a result of the gel electrophoresis represents the marking, similarly to a bar code. The band sequence can also be translated into a number.
The previously mentioned processes are disadvantageous in a number of respects:
a) The processes are time- and cost-consuming, because for the identification of the marking, a PCR and a gel electrophoresis have to be carried out;
b) the processes are difficult to automate, because the reaction products formed in the PCR have to be transferred to a gel for carrying out the gel electrophoresis;
c) the processes are error-prone, because a large number of contamination-sensitive pipetting operations are necessary to carry them out;
d) the processes are laborious because, for marking a large number of substances, the same number of labeling DNAs have to be prepared.
A further process is disclosed in U.S. Pat. No. 5,139,812. Here, an ink containing [lacuna] specified nucleic acid sequence is used for the falsification-proof marking of articles. In order to mark a plurality of articles in a distinguishable manner, different inscriptions are applied using the ink. For the identification of a marking applied in such a way, the inscription is rendered visible by means of a color reaction. Identification can also be carried out by a radioactive marking of the nucleic acid sequence used. This process is therefore especially disadvantageous, because the application of a distinguishable marking is troublesome and the marked article has to be destroyed for identification.
It is furthermore disclosed in WO 95/17737 to use the artist's blood for marking works of art. For identification, the marking is compared by means of DNA analysis with a deposited further blood sample. This process does not make possible the preparation of a large number of different markings without problems. Identification is laborious.
The object of the invention is to indicate a process and a kit for carrying it out with which a large number of substances or articles can be marked in a falsification-proof and distinguishable manner and can subsequently be identified in an inexpensively and rapidly. This object is achieved by the features of claims
1
and
20
. Expedient embodiments of the invention result from the features of claims
2
to
19
and
21
to
27
.
According to the achievement of the invention as regards the process, it is proposed that, for identification, a first primer group having a first primer section corresponding to the first sequence section and a second primer group having a third primer section corresponding to the third sequence section is used, where each of the primer groups comprises four primer variants, in each case differing in at least one additional terminally provided base, such that just one primer variant of the first primer group together with just one primer variant of the second primer group makes possible amplification of the nucleic acid sequence.
Using the process according to the invention, a distinguishable marking or internal numbering of a large number of articles can be carried out simply. Using a relatively small number of different marking or nucleic acid sequences, a large number of different markings can be made available. The markings are identifiable rapidly and without great expenditure.
According to one embodiment of the invention, the nucleic acid sequence has more than 20, preferably 40, nucleotides.
Nucleic acid sequences which differ in the second sequence section are advantageously used for marking. This advantageously consists of two part regions, of which each is occupied by at least one base. The combination of the two part regions makes possible the preparation of a total of 16 different nucleic acid variants, namely adenine (=A)−A, A-thymidine (=T), A-guanine (=G), A-cytosine (=C), T-A, T-T, T-G, T-C, G-A, G-T, G-G, G-C, C-A, C-T, C-G, C-C. The 16 nucleic acid variants form a nucleic acid set.
Advantageously, the second sequence section is formed of two part regions, of which at least one consists of a plurality of bases. Accordingly, the corresponding primer variant is then terminally provided with the same number of corresponding bases. As a result, the probability of the formation of false-positive samples is drastically reduced. The part region can consist of a specific sequence or of a number of identical bases.
To increase the combination possibilities, further nucleic acid sets can be used whose nucleic acid variants differ in the first and third sequence section. For example, to increase the combination possibilities from 16 to 16
2
, the previously described nucleic acid variants of a nucleic acid set can be combined with further nucleic acid variants which are selected from a second nucleic acid set. The second nucleic acid set differs from the first nucleic acid set in the first and third sequence section. Analogously, the coding capacity can be increased to 16
n
by the use of n nucleic acid sets.
The first and the third sequence section preferably have the same number of nucleotides. The first and the third sequence section are expediently constructed such that they are fusible under comparable stringency conditions. This makes possible a very simple amplification by means of polymerase chain reaction (PCR) or ligase chain reaction (LCR).
The nucleic acid sequences used can, of course, also be nucleic acid derivative sequences, in particular protein-like nucleic acid (PNA) or phosphortionate [sic] nucleic acids (PTO) or hybrids thereof. Nucleic acid derivative sequences of this type are in some cases distinguished by improved stability.
To protect one or more nucleic acid sequence(s) applied to solid articles, these can be covered by a protective layer, such as wax. The nucleic acid sequence(s) can furthermore be a constituent of a layer applied to solid articles, such as a lacquer. Marking can also be brought about by impregnation or mixing.
For identification of the at least one nucleic acid sequence, this is expediently extracted and a solution containing the nucleic acid sequence is prepared. The nucleic acid sequence can then be duplicated by means of PCR or LCR using the primer variants. The duplicated nucleic acid sequence or the amplificate is expediently subsequently detected by means of fluorescence, DNA, gel electrophoresis, restriction analysis, hybridization or by means of sequencing. Detection by means of fluorescence is particularly simple and quick to carry out. It can take place directly in a microtiter plate.
According to the invention, to carry out the process, a kit for the identification of a marking [lacuna], where the kit has a first primer group having a first primer section corresponding to the first sequence section and a second primer group having a third primer section corresponding to the third sequence section, where each of the primer groups comprises four primer variants, in each case differing in at least one additional terminally provided base, such that just one primer variant of the first primer group together with just one primer variant of the second primer group is complementary to the nu
Bertling Wolf
Kosak Hans
Fish & Richardson P.C. P.A.
Jones W. Gary
november Aktiengesellschaft Gesellschaft fur Molekulare Medizin
Taylor Janell E.
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